红芪总黄酮对离心运动大鼠骨骼肌细胞NF-κB信号通路相关凋亡蛋白表达的影响

Effects of Total Flavonoids of Hongqi(Hedysarum Polybotry s)on NF-κB Signal Pathway Related Apoptotic Proteins in Skeletal Muscle Cells of Rats after Eccentric Exercise

目的:探讨红芪总黄酮对离心运动大鼠骨骼肌细胞NF-κB信号通路相关凋亡蛋白表达的影响。方法:健康雄性Wistar大鼠随机分为3组:安静对照组(CG)、离心运动组(EG)和离心运动+药物预处理组(DPG),每组8只。DPG组在离心运动前3 d给予红芪总黄酮灌胃,CG组、EG大鼠给予等量生理盐水灌胃。EG组、DPG组大鼠均采用恒定速度20 m/min,-16°下坡跑台离心运动,持续运动至力竭。RT-qPCR法检测骨骼肌细胞核转录因子kappa B(nuclear factor kappa-B,NF-κB)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,bcl-2)、Bcl-2相关X基因(Bcl-2 Associated X Protein,bax)、细胞色素C(cyt c)、半胱氨酸蛋白酶-3(caspase-3)mRNA表达的影响,Western blotting法检测骨骼肌细胞pNF-κB、Bcl-2、Bax、Cyt c、Caspase-3蛋白的表达,TUNEL法检测骨骼肌细胞凋亡率。结果:EG组和DPG组NF-κB、Bax、Cyt c、Caspase-3 mRNA和蛋白的表达明显高于CG组(P<0.05),DPG组及EG组的Bcl-2 mRNA和蛋白表达显著低于CG组(P<0.05),Bcl-2/Bax比值也显著低于CG组(P<0.05);与EG组比较,DPG组NF-κB、Bax、Cyt c、Caspase-3 mRNA和蛋白的表达明显降低(P<0.05),DGP组的Bcl-2 mRNA和蛋白表达显著高于EG组(P<0.05),DGP组Bcl-2/Bax比值较EG组也显著增高(P<0.05)。TUNEL染色结果显示,与CG组比较,EG组、DPG组大鼠骨骼肌细胞凋亡指数显著升高(P<0.01);与EG组比较,DPG组大鼠骨骼肌细胞凋亡指数明显降低(P<0.05)。结论:一次离心力竭运动可激活大鼠骨骼肌细胞NF-κB信号分子,启动细胞的凋亡程序;红芪总黄酮能够降低高强度运动引发的应激反应,从而延缓离心运动后骨骼肌疲劳和损伤的发生,其机制可能与降低骨骼肌组织NF-κB、Bcl-2/Bax、Cyt c、Caspase-3表达水平、抑制骨骼肌细胞凋亡有关。

Objective:To study the effects of total flavonoids of Hedysarum Polybotrys on NF-κB signal pathway related apoptotic proteins in skeletal muscle of rats after eccentric exercise.Methods:Healthy male Wistar rats were randomly divider into three groups:control group(CG),an eccentric exercise group(EG),a drug pretreatment plus eccentric exercise group(DPG),8 rats in each group.Rats in the drug pretreatment group plus eccentric exercise group were administered total flavonoids of Hedysarum Polybotrys(0.2 mg/kg)orally on 3 days before the centrifugal movement,and the rats in the other groups were given the same amount of normal saline,once a day.EG and DPG group were submitted to a single bout of eccentric exercise at 20 m/min on a treadmill down a 16°decline,continuous trainning to exhaustion.Gastrocnemius muscle of rats in all groups were obtained.The expressions of NF-κB,bcl-2,bax,cyt c and caspase-3 mRNA in skeletal muscle cells were determined by real-time fluorescence quantitative RT-qPCR.Western blotting was used to detect the NF-κB,Bcl-2,Bax,Cyt c,Caspase-3 protein of Skeletal muscle tissue.Cells of skeletal muscle apoptosis was detected using terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)method.Results:Compared with CG group,the expressions of NF-B,Bax,Cyt c,Caspase-3 mRNA and protein in EG group and DPG group were significantly increased(P<0.05),while the expressions of Bcl-2 mRNA and protein were significantly decreased(P<0.05),and the Bcl-2/Bax ratio was significantly decreased(P<0.05).Compared with the EG group,the expressions of NF-B,Bax,Cyt c,Caspase-3 mRNA and protein in the DPG group were significantly decreased(P<0.05),while the expressions of Bcl-2 mRNA and protein were significantly increased(P<0.05),and the Bcl-2/Bax ratio was significantly increased(P<0.05).The TUNEL staining results showed that the apoptosis index of skeletal muscle cells in EG and DPG group was significantly increased(P<0.01)compared with CG group,Compared with EG group,the apoptosis index of skeletal muscle cells in DPG group was significantly reduced(P<0.05).Conclusion:The activation of NF-κB in skeletal muscle cells after eccentric exercise can activate the cell apoptosis process.Total flavonoids of Hedysarum Polybotrys can reduce oxidative stress after high intensity exercise,delay the incidence of skeletal muscle fatigue and injury.The mechanism may be related to the decreased NF-κB,Bcl-2/Bax,Cyt c and Caspase 3 in skeletal muscle tissues and inhibited skeletal muscle cell apoptosis.